Ablation based laser machining of biomolecule patterns on substrates

ABSTRACT

A method for patterning a one or more biomolecules on a substrate that includes coating the substrate with a coating of the one or more biomolecules, applying a laser to the coating, and ablating a portion of the one or more biomolecules with the laser in a predetermined pattern.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation-in-Part of international patentapplication no. PCT/US2007/069932, filed May 30, 2007, and aContinuation-in-Part of U.S. patent application Ser. No. 11/755,187,filed May 30, 2007, both of which claim priority to U.S. provisionalpatent application No. 60/809,625, filed May 31, 2006, the contents ofwhich applications are incorporated herein by reference in theirentireties.

BACKGROUND

Methods and compositions for patterning biological material on asubstrate are described, including a method for ablating biomolecules ona substrate with a laser to form a pattern. Patterned substrates andapplications thereof are also described.

Control of the position or distribution of biologically active moleculesor biomolecules on a substrate is important for a wide range ofscientific and technological applications. This controlled positioninghas commonly become known as “patterning” of these molecules. Forexample, the patterning of DNA oligonucleotides on a glass substrate isused to make microarrays; similarly the patterning of proteins on asubstrate is used to make protein arrays. These types of arrays have arange of analytical and diagnostic applications. In cell biology,substrates patterned with extracellular matrix proteins are used tocontrol the shape, position and behavior of cells.

To pattern biologically active molecules, a wide range of methods havebeen developed. These methods can be divided into two classes:self-assembled patterning and directed patterning. In self-assembledpatterning the physical chemical properties of a molecule or combinationof molecules are exploited under specific conditions to producedistributions of molecules with known non-random organizationalproperties. For example, a self-assembled monolayer of alkanethiols ongold will often have a high degree of order that results fromintermolecular interactions between the components of the molecules.This order is a pattern, and it in turn can be used to create patternsof other molecules. Also, colloidal particles adsorbed onto a surfacecan have varying degrees of order that can be used to create patterns.

In directed patterning, the position of molecules is controlled byinformation that is brought in from the outside, such as a mask or atemplate. Directed patterning methods can in turn be divided into twotypes: lithographic approaches and writing approaches. Lithographicapproaches include methods where a physical template such as a mask or amold is used to transfer a pattern to an object. Examples includeconventional photolithography and microcontact printing. In contrast,writing approaches use a serial approach to transfer a pattern,typically from a computer-based representation such as a CAD (computerassisted design) drawing, to an object. Electron beam lithography,despite its name, is a writing approach, by the definition used here. Ingeneral, lithographic approaches are good for producing many copies ofthe same pattern; writing approaches are good for producing uniquepatterns for producing a large number of different patterns, or forchanging patterns quickly.

As noted above, there are a wide range of applications for patterningproteins and other biomolecules. One area that has become increasinglyimportant over the past decade is patterning of proteins for cellbiological and related applications. In vitro cell culture was developedto facilitate the study of the biomedical and industrial uses of cellsoutside of an animal. During the last century cell culture techniquesand materials have been refined to more accurately reflect the in vivoenvironment of the cells, to provide analytically and diagnosticallyinformative responses from cells, and to more efficiently grow cells forresearch and industrial biomedical uses. One aspect of the cellularmicroenvironment that is not well-captured by traditional cell cultureis the spatial heterogeneity of molecules in the extracellularenvironment. Molecules are not randomly arranged or uniformly arrangedin the extracellular environment, and the spatial organization of themolecules influences cell structure and function.

SUMMARY

A method for patterning one or more biomolecules on a substrate includescoating the substrate with the one or more biomolecules; applying alaser onto the one or more of molecules; and ablating a portion of theone or more biomolecules with the laser in a predetermined pattern. Thepredetermined pattern has one or more ablated portions and one or morenon-ablated portions on the substrate. The one or more ablated portionshas less than 100% of biological function or activity, or biochemicalfunction or activity of the one or more biomolecules on the substrate.The one or more non-ablated portions has one or more active orfunctional biomolecules of the one or more biomolecules on thesubstrate.

Ablating may include inactivating or rendering non-functional at leastone of the one or more molecules with the laser such that the at leastone of the one or more molecules remains on the substrate, removing oneor more of the one or more biomolecules with the laser, breaking atleast one covalent bond of the one or more biomolecules, changing aconformation of the one or more biomolecules, changing an orientation ofthe one or more biomolecules, or any combination thereof.

Applying the laser may include translating the substrate in one, two, orthree dimensions to form the predetermined pattern, applying the laserfurther includes rotating the substrate around one, two, or three axesto form the predetermined pattern, or translating the substrate in one,two, or three dimensions and rotating the substrate around one, two, orthree axes to form the predetermined pattern. Applying the laser mayinclude directing electromagnetic radiation produced by the laser to thesubstrate via an optical system that includes, for example, lenses,mirrors, filters, shutters, polarizers, and any other optical devicethat can modify the electromagnetic radiation or its path through space,and any combination thereof. The electromagnetic radiation may includecontinuous wave radiation or pulsed radiation. Applying the laser mayinclude modulating the power, the pulse width, the pulse frequency, theirradiance, the fluence, and any combination thereof of theelectromagnetic radiation arriving at the substrate.

The one or more biomolecules may include proteins, peptides, nucleicacids, drugs, lipids, bioactive polymers, bioactive compounds, and anycombination thereof. The one or more biomolecules that are proteins mayinclude fibronectin, vitronectin, collagen, growth factors, cellularmembrane proteins, intracellular proteins, extracellular matrixproteins, soluble proteins, signaling proteins, and any combinationthereof. The one or more of biomolecules may be attached to a particleor colloid including, for example, quantum dots, superparamagneticnanoparticles, dendrimers, glass or silica particles, liposomes, virusesor phage particles and analogous particles, and any combination thereof.

The substrate may include glass, polymeric material, silicon, plastic,rubber, metal, ceramic, any material that is not substantially destroyedor damaged by the laser, and any combination thereof. The material thatis not substantially destroyed or damaged may include material that isnot substantially destroyed or damaged when laser irradiation of an areaof a substrate removes material to a depth of more than 1 nanometer,more than 3 nanometers, more than 5 nanometers, more than 10 nanometers,more than 25 nanometers, more than 50 nanometers, more than 100nanometers, more than 500 nanometers, more than 1 micrometer, more than5 micrometers, more than 10 micrometers, more than 50 micrometers, ormore than 100 micrometer from a point in an area irradiated by thelaser.

Coating the substrate may include adsorbing the one or more biomoleculeson the substrate. Adsorbing the one or more biomolecules on thesubstrate may include covalently attaching to the substrate,electrostatically attaching to the substrate, hydrophobically attachingto the substrate, sterically attaching to the substrate, entropicallyattaching to the substrate, and any combination thereof. Coating thesubstrate may include one or more biomolecules covalently ornon-convalently attached to the substrate via a coupling molecule ofvarying length. Coating the substrate may include coating a non-uniformsubstrate.

The laser may remove the at least one of the one or more biomolecules ata first dose and inactivate at least one of the one or more biomoleculesat a second dose, and wherein the second dose is lower than the firstdose. Ablating may include forming a microplasma and applying themicroplasma to the portion of the one or more biomolecules, patterning aportion of the one or more biomolecules of one type of biomolecule in acombination of two or more types of biomolecules, transferring heat fromthe laser to the substrate, a solvent or medium above the substrate, atleast one of the one or more biomolecules, and any combination thereof.The predetermined pattern may contain a gradient of inactivatedbiomolecules, a gradient of removed biomolecules, or a combination ofgradients of inactivated and removed biomolecules. The gradient mayinclude a change in an amount of activity or function of the one or morebiomolecules per unit distance, unit area, or unit volume as a functionof position on the substrate, and any combination thereof.

The substrate may be translucent, where applying the laser includesapplying the laser through an exterior side of the substrate onto anopposing interior side of the substrate, wherein the interior side ofthe substrate has the one or more biomolecules applied thereon, andwherein said molecules are partially or fully ablated in thepredetermined pattern.

The method may include backfilling the one or more ablated portions witha second one or more biomolecules.

The one or more biomolecules may be hydrated in a liquid or dry layer.The one or more biomolecules may be in an aqueous solvent environment orin a non-aqueous solvent environment.

A substrate coated with one or more biomolecules in a pattern includes afirst portion on the substrate having a coating. The coating has the oneor more biomolecules and one or more ablated portions on the substrate.The one or more ablated portions has at least one dimension in the planeof the substrate from about at least 0.1 nanometer, at least 1nanometer, at least 10 nanometers, at least 100 nanometers, or at least250 nanometers to 1 meter. One or more non-ablated portions on thesubstrate has at least one lateral dimension in the plane of thesubstrate from about at least 0.1 nanometer, at least 1 nanometer, atleast 10 nanometers, at least 100 nanometers, or at least 250 nanometersto 1 meter (lateral dimension). The one or more ablated portions haveless than 100% of biological function or activity, or biochemicalfunction or activity of the one or more biomolecules on the substrate.

The substrate coated with the one or more biomolecules may be amicroscope coverslip, microscope slide, petri dish, tissue cultureflask, biomedical implant, test tube, eppendorf tube, diagnostic assay,a biochip, a protein/nucleic acid biochip sensor, a cell-based sensor, alab-on-a-chip assay, a lab-in-a-capillary assay, a cell adhesion assay,a cell translocation/migration/invasion/chemotaxis assay, or aneuronal-guidance assay. The substrate may be non-flat. The one or moreablated portions have inactivated or non-functional biomolecules. Theone or more ablated portions may include at least one of the one or morebiomolecules having a broken covalent bond, having been removed, havinga changed conformation, and having a changed orientation, and anycombination thereof. The substrate may have an exterior side opposite aninterior side, and wherein the interior side has the coating appliedthereon. The one or more biomolecules may be hydrated by a thin liquidor dry layer. The one or more biomolecules may be in an aqueousenvironment or a non-aqueous environment. The one or more biomoleculesinclude, for example, proteins, peptides, nucleic acids, drugs, lipids,bioactive polymers, bioactive compounds, and any combination thereof.One or more biomolecules may be covalently or non-convalently attachedto the substrate via a coupling molecule of varying length. The one ormore biomolecules may be attachable to the substrate and are sensitiveto laser exposure. The one or more of protein biomolecules may includefibronectin, vitronectin, collagen, growth factors, cellular membraneproteins, intracellular proteins, extracellular matrix proteins, solubleproteins, signaling proteins, and any combination thereof. The one ormore of biomolecules may be attached to a particle or colloid includequantum dots, superparamagnetic nanoparticles, dendrimers, glass orsilica particles, liposomes, viruses or phage particles and analogousparticles, and any combination thereof. The substrate may includesilicon, plastic, rubber, metal, ceramic material, or any material thatis not destroyed or damaged by the laser, and any combination thereof,wherein the material that is not substantially destroyed or damagedincludes material that is not substantially destroyed or damaged whenlaser irradiation of an area of a substrate removes material to a depthof more than 1 nanometer, more than 3 nanometers, more than 5nanometers, more than 10 nanometers, more than 25 nanometers, more than50 nanometers, more than 100 nanometers, more than 500 nanometers, morethan 1 micrometer, more than 5 micrometers, more than 10 micrometers,more than 50 micrometers, or more than 100 micrometers from a point inan area irradiated by the laser.

A patterned substrate coated with a one or more biomolecules includes apattern obtainable by coating the substrate with the one or morebiomolecules; applying a laser onto the one or more of molecules; andablating a portion of the one or more biomolecules with the laser in apredetermined pattern. The predetermined pattern has one or more ablatedportions and one or more non-ablated portions on the substrate. The oneor more ablated portions has less than 100% of biological function oractivity, or biochemical function or activity of the one or morebiomolecules on the substrate. The one or more non-ablated portions hasone or more active or functional biomolecules of the one or morebiomolecules on the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a side view of an illustrative substrate in accordance withthe present disclosure;

FIG. 1B is a side view of an illustrative biomolecule coated substrate;

FIG. 1C is a schematic diagram of a laser system applying a laser to abiomolecule coated substrate;

FIG. 1D is a schematic diagram of a laser system translating andshuttering a beam onto a biomolecule coated substrate to form a pattern;

FIG. 1E is a side view of a surface having an arbitrary pattern ofablated biomolecules and non-ablated biomolecules on a surface and planview of the surface having the arbitrary pattern of ablated biomoleculesand non-ablated biomolecules on the surface;

FIG. 1F is a side view of cells grown on a surface, a plan view of cellsgrown on a surface within different shapes of ablated biomolecules andnon-ablated biomolecules, and a plan view immunofluorescence lightmicrograph of cells grown within a rectangular pattern, where the activefibronectin is stained with an antibody against fibronectin, and thecells are stained with a non-specific membrane label. The fluorescenceintensity of the fibronectin staining is proportional to the activity ofthe fibronectin; thus, the fibronectin activity in the pattern rangesfrom high (light areas) to low (dark areas);

FIG. 2A is a cross-sectional view of a translucent tube having solventpresent within an interior of the translucent tube, a front perspectiveview of the translucent tube having solvent present within the interior,and a side view of the translucent tube having solvent present withinthe interior;

FIG. 2B is a cross-sectional view of biomolecules coated on an interiorsurface of a translucent tube in the presence of solvent and a side viewof the biomolecules coated on an interior surface of a translucent tubein the presence of solvent;

FIG. 2C is a schematic diagram of ablating biomolecules on an interiorsurface of a translucent tube in the presence of solvent with a lasersystem and a side view of ablated and non-ablated biomolecules on theinterior surface of the translucent tube in the presence of solvent;

FIG. 2D is a schematic diagram of translating a laser with a mirror,activating and deactivating the laser with a shutter, and translatingand/or rotating the translucent tube, and a side view of ablated andnon-ablated biomolecules on an inside surface of the translucent tube inthe presence of solvent;

FIG. 2E is a schematic diagram of applying a laser to predeterminedareas of biomolecules while translating the laser with a mirror,activating and deactivating the laser with a shutter, and rotatingand/or translating the translucent tube forming patterned biomoleculeson an inside surface of the translucent tube, a side view of ablated andnon-ablated biomolecules on the inside surface of the translucent tubein the presence of solvent in a pattern, and cultured adherent cells onthe inside surface of the translucent tube that are influenced by thepatterned biomolecules;

FIG. 3A is an immunofluorescence image of a surface coated withsurface-bound fibronectin, where the fibronectin is stained with anantibody against fibronectin in a pattern formed by different doses of alaser in accordance with the present disclosure showing dose dependenceof inactivation of surface-bound fibronectin with the dose of the laserincreasing from left to right, and the fluorescence intensity of thefibronectin staining is proportional to the activity of the fibronectin;thus, the fibronectin activity in the pattern ranges from high (lightareas) to low (dark areas), the image is 225 micrometers wide;

FIG. 3B is a graph showing normalized fibronectin activity, as measuredby the fluorescence resulting from the antibody against fibronectin as afunction of applied laser dose, on the surface of FIG. 3A;

FIG. 4A is an atomic force microscope topographic image of a surfacecoated with surface-bound fibronectin, having a pattern in which thevertical lines have been exposed to the laser one, five, and nine timesas illustrated in the inset, the image is 90 micrometers wide;

FIG. 4B is an immunofluorescence image of the image of FIG. 4A, wherefollowing a topographic imaging for FIG. 4A, the fibronectin is stainedwith an antibody against fibronectin, and the fluorescence intensity ofthe fibronectin staining is proportional to the activity of thefibronectin; thus, the fibronectin activity in the pattern ranges fromhigh (light areas) to low (dark areas); the image is 120 micrometerswide;

FIG. 4C is a graph showing a first plot of fluorescence intensity (thickline) as a function of position from left to right within a dotted boxof FIG. 4B and showing a second plot (thin line) showing height fromleft to right within the dotted box of FIG. 4A showing profiles ofatomic force microscope topograph (thin line) demonstrating thatinactivation can be achieved without removal of mass that is measurableby the atomic force microscope;

FIG. 5 is an immunofluorescence image of a surface coated withsurface-bound fibronectin, where the fibronectin is stained with anantibody against fibronectin, in a pattern formed by different doses ofa laser in accordance with the present disclosure showing dosedependence of inactivation of surface-bound fibronectin with the doseincreasing from left to right, and where biomolecules on the substratewere submerged in an aqueous solution when ablation was performed, andthe fluorescence intensity of the fibronectin staining is proportionalto the activity of the fibronectin; thus, the fibronectin activity inthe pattern ranges from high (light areas) to low (dark areas), and theirregular dark lines and shapes are the result of abrasion of the samplepost laser exposure;

FIG. 6A is an immunofluorescence image of a surface coated withsurface-bound fibronectin, where the fibronectin is stained with anantibody against fibronectin, in a pattern formed by point exposures ofa CO₂ laser with a wavelength of 10.6 μm, and the scale bar represents35 micrometers, and the fluorescence intensity of the fibronectinstaining is proportional to the activity of the fibronectin; thus, thefibronectin activity in the pattern ranges from high (light areas) tolow (dark areas);

FIG. 6B is a graph showing a plot of fluorescence intensity as afunction of position from left to right within the dashed line in FIG.6A;

FIG. 7A is an immunofluorescence image of a surface coated withsurface-bound fibronectin, where the fibronectin is stained with anantibody against fibronectin, in a pattern formed by a single pointexposure of a YAG (yttrium aluminium garnet) laser with a wavelength of1064 nanometers, and the scale bar represents 35 micrometers, and thefluorescence intensity of the fibronectin staining is proportional tothe activity of the fibronectin; thus, the fibronectin activity in thepattern ranges from high (light areas) to low (dark areas);

FIG. 7B is a graph showing a plot of fluorescence intensity as afunction of position from left to right within the dashed line in FIG.7A;

FIG. 8. Patterning inside a capillary tube.

FIG. 8A. Photograph of the capillary.

FIG. 8B. Low magnification fluorescence image.

FIG. 8C-F. Lines patterned in a fibronectin coating on the inside of thetube, in two orientations and with the focus at the center or edges ofthe tube;

FIG. 9. AFM characterization of inactivated regions. 9A.Immunofluorescence image (upper) and 9B. AFM height image (lower) ofthree lines patterned by laser exposure. The laser exposure inactivatedprotein without removing it from the surface or otherwise modifying thetopography. Active protein is light, inactivated protein is dark. Thefull z-range of the AFM image is 50 nm, and thus any topography greaterthan ˜0.5 nm would be visible. The dark square in the fluorescence imageis the result of letting the AFM tip drift into the sample during anovernight run and effectively rubbing away protein. The image shown wascollected during the first scans, prior to the removal of the protein;

FIG. 10. Inactivation patterning of fibronectin on common cell culturesubstrates detected by immunofluorescence microscopy. Active protein islight, inactivated protein is dark. 10A. Silicon. 10B. Quartz. 10C.Glass. 10D. Polystyrene. 10E. PDMS. Scale bars=35 Mm;

FIG. 11. Inactivation patterning of ECM proteins on glass. Activeprotein is light, inactivated protein is dark. 11A. Collagen. 11B.Laminin. Scale bars=35 μm;

FIG. 12. Laser-inactivated protein patterns support the formation offocal adhesions. Fluorescence microscopy image of a fibroblastinteracting with patterned fibronectin. 12A. Fibronectin. Activefibronectin is light, inactive fibronectin is dark, 12B. Actin, 12C.Vinculin, a protein found in focal adhesion complexes, 12D. Compositeimage of fibronectin, actin, and vinculin. Focal adhesions areidentified with arrows.

FIG. 13. Gradient patterns of fibronectin on glass substrates.Immunofluoresence images of gradient patterns of fibronectin. Activeprotein is light, inactivated protein is dark. 13A. Near linear. 13B.Logrithmic. 13C. Curved linear. Scale bars, 32 μm;

FIG. 14. Schematic of an 18×24 inch stage above which a laser istranslated to pattern objects placed on the stages surface. Threeone-inch coverslips coated with fibronectin are shown on the stage.These where patterned by laser ablation, where the patterning wascarried out from a single input pattern, without moving the coverslipsduring the patterning, and without interrupting from movement of thelaser after the patterning commenced. The coverslips were subsequentlystained with an antibody for fibronectin and visualized by fluorescencemicroscopy. The patterns are well formed and agree with the inputpatterns to the computer-controlled stage. Differences in rotationbetween the patterns are due to differences in how the round coverslipsare mounted in the fluorescence microscope for imaging. Note that thelaser used here has a relatively large spot size (˜50 μm diameter), andhas an ˜10 μm wavelength.

FIG. 15. Gabor expansion of gradient functions into sums of Gaussians.Arbitrary functions ƒ(x) (clockwise from top left (FIGS. 15A-D), ƒ(x)=c,x, e^(x), ln(x)) can be decomposed into sums of Gaussians with fixed σ.The input function ƒ(x) is represented with ⋄, the g(x) are Gaussiantraces at the bottom of the plots, and the sum of the Gaussians is theupper line. A total of 100 Gaussians were summed. Δx=σ.

FIG. 16. Gabor expansion using modified Gaussians. A. A functionp_(w)(x) with Gaussian sides and a plateau of width w. B. The scalefactor s(w/σ) used to compensate for the plateau is proportional to w/σ.C-1 to C-4. Arbitrary functions (clockwise from top left,ƒ(x) =c, x,e^(x), ln(x)) decomposed into sums of p_(w)(x). The input function ƒ(x)is represented with ⋄, the p_(w)(x) are the traces on the lower portionof the plots, and the sum of the p_(w)(x) is the upper line. A total of100 p_(w)(x) were summed. Δx=σ.

DETAILED DESCRIPTION

While the present disclosure may be susceptible to embodiment indifferent forms, there are shown in the drawings, and herein will bedescribed in detail, embodiments with the understanding that the presentdescription is to be considered an exemplification of the principles ofthe disclosure and is not intended to limit the disclosure to thedetails of construction and the arrangements of components set forth inthe following description or illustrated in the drawings.

Referring now to FIGS. 1A through 1F, a patterned substrate and a methodfor ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are shown. Ablating removes or inactivatesactive portions or functional portions of biomolecules. Ablation caninclude a change in conformation of a biomolecule, a change inorientation of a biomolecule, a breaking of at least one covalent bondin a biomolecule, a removal at least one atom from one or morebiomolecules and at most 100% of the one or more biomolecules, such thatan activity or a function of the biomolecule or group of biomolecules ispartially or fully lost. Biomolecules are any biologically activemolecules or molecules that modify a function or an activity of abiological or biochemical entity.

As shown in FIG. 1A, a substrate 10 is shown. Substrate 10, for example,is glass, polymeric material, silicon, plastic, rubber, metal, ceramicmaterial, and any material that is not destroyed or substantiallydamaged by laser irradiation, or any combination thereof. Substantialdamage occurs when laser irradiation of an area of substrate 10 removesmaterial to a depth of more than 1 nanometer (nm), more than 3 nm, morethan 5 nm, more than 10 nm, more than 25 nm, more than 50 nm, more than100 nm, more than 500 nm, more than 1 μm, more than 5 micrometer (μm),more than 10 μm, more than 50 μm, or more than 100 μm from some point inthe area irradiated by the laser.

Substrate 10 may be a non-flat substrate or a three dimensionalsubstrate selected from the group consisting of for example, amicrofabricated substrate or device, Micro-Electro-Mechanical Systems(MEMS) devices, a Petri dish, a vascular stent, an auditory implant, andanalogous devices. Substrate 10 may be an interior substrate or opposingside of a translucent material. Substrate 10 may be an interiorsubstrate of a translucent material of a three-dimensional structure.Substrate 10 can be an interior substrate of a solid, semi-solid, orgel-like translucent material that is defined by relative translation oflaser 20 to the material.

Substrate 10 has a coating of one or more active or functionalbiomolecules 12 applied thereon, as shown in FIG. 1B. Biomolecules areany biologically active molecules or molecules that modify a function oran activity of a biological or biochemical entity. One or morebiomolecules 12 are biomolecules that are attachable to a substrate andcan be ablated by laser exposure. One or more biomolecules 12 may be anybiomolecule that can be attached, covalently or noncovalently to asubstrate. One or more biomolecules can be attached directly to thesubstrate or via a molecular or polymeric tether. One or morebiomolecules 12, for example, may include proteins; extracellular matrixproteins such as fibronectin, vitronectin and collagen; soluble proteinssuch as growth factors, including vascular endothelial growth factor(VEGF), brain-derived neurotrophic factor (BDNF) or neuronal growthfactor (NGF); proteins that are part of a cellular membrane, such assemaphorins, neuropilins, PAR1 receptor, ephrins or plexins; proteinsthat are intracellular; a coupling biomolecule that links a protein orother biomolecule to a substrate such as streptavidin or antibodies;blocking agents to provide space for coupling biomolecules, proteins orother biomolecules to bind to a substrate; antibodies; receptors;ligands; lipids; antigens; full-size proteins; protein domains;peptides; enzymes and/or enzyme substrates; polysaccharides; DNA, RNA,or other nucleic acids; small biomolecules such as nucleotides (e.g.cyclic adenosine monophosphate); fluorescent reporters, small moleculesand drugs, peptides and enzymatic substrates; small molecules that bindcovalently to proteins, peptides or nucleic acids; aggregates ofbiomolecules, small particles or colloids less than 10 μm, less than 5μm, less than 1 μm, less than 500 nm, less than 200 nm, less than 100nm, or less than 50 nm diameter, including quantum dots,superparamagnetic nanoparticles, quantum dots coated with biomoleculesas described above, superparamagnetic nanoparticles coated withbiomolecules as described above, dendrimers coated with biomolecules asdescribed above, glass or silica particles coated with biomolecules asdescribed above, liposomes coated with biomolecules as described above,viruses or phage particles and analogous particles; or any combinationthereof.

The coating of one or more biomolecules 12 may be by any one of manyconventional methods. The coated biomolecule may be covalently bound, ornon-covalently bound. Covalent chemistries may include couplingcompounds of the general structure X—R—Y, where X contains at least onereactive group that covalently binds to the substrate and Y contains atleast one reactive group that covalently binds to the biomolecule, and Ris a spacer group of varying length that does not react with eithersubstrate or biomolecule. Examples of X and Y include components such assilanols, silanes, thiols, esters, gluteraldehyde, NHS-ester, DSP, otherthiol-terminated compounds, cysteine residues, carboxylate treated withcarbodiimide, and any combination thereof. Non-covalent chemistriesinclude adsorption to properly modified glass or other substrate, wherethe attachment of the molecule may be mediated by electrostatic forces,van der Waals forces, hydrophobic interactions, physical trapping,capillary forces, entropic forces and interactions, steric interactions,and any combination thereof.

One or more molecules 12 coated on substrate 10 may be have a thickness13 of about 1 nm to about 20 nm. Thickness 13 of one or morebiomolecules 12 may be a layer that is on average more than about 0 nmthick and less than about 50 μm, less than about 10 μm, less than about5 μm, less than about 1 μm, less than about 500 nm, less than about 200nm, less than about 100 nm, less than about 50 nm, less than about 20nm, less than about 10 nm, less than about 5 nm, or less than about 1 nmthick. One or more molecules 12 coated on substrate 10 can be uniform ornon-uniform. A non-uniform coating may be a coating including one ormore biomolecules that are patterned, for example, by the methoddescribed herein or another method of patterning on substrate 10.

One example of substrate 10 coated with one or more biomolecules 12includes coating fibronectin on glass coverslip substrates of dimensions22 mm×22 mm. The coverslip substrates are plasma cleaned forapproximately 5 minutes with a plasma cleaner (Harrick Plasma, Ithaca,N.Y.). The coverslip substrates are then cleaned for about 20 minutes inpirahna-solution, H₂O₂:H₂SO₄ (1:3 v/v). The coverslip substrates arethen washed in water. The coverslip substrates are incubated in anethanolic 3-aminopropyltriethoxysilane (APTES); Gelest, Inc.,Morrisville, solution, ethanol 99.5%/APTES 99+%, 10:1 v/v solution, in 1milliMolar (mM) acetic acid for 3 hours, rinsed in water, then driedovernight in a dry-box having approximately <25% relative humidity orbaked at 110° C. for about 2 to about 3 hours. Approximately 200microliters (μL) of human fibronectin (Invitrogen, Carlsbad, Calif.) inphosphate buffered saline (PBS) is pipetted onto the coverslipsubstrates. The coverslip substrates are incubated at 4° C. for about 12to about 16 hours and then at 37° C. for about 2 hours. The coverslipsubstrates are then rinsed in water and dried under nitrogen gas. Thecoverslip substrates are stored in a drybox and may be used within threeweeks although the fibronectin retains functions for at least severalmonths.

A laser 20 is applied to the coating of one or more biomolecules 12, asshown in FIG. 1C. Laser 20 may include a laser system having a focusinglens system 22, a mirror 24, and/or shutter system 26. Laser 20 appliesa dose of electromagnetic radiation sufficient to cause a predeterminedlevel of ablation on substrate 10. The amount of laser radiationdelivered to the biomolecules on the surface can be described by theapplied dose of the laser. There are two components to the dose of thelaser—the intensity of the laser on the biomolecules and the total timethe biomolecules are exposed to the laser. The intensity, also calledpower density and irradiance, of the laser is the amount of energy perunit time per unit area the laser delivers to the substrate. Theintensity depends on the optical system of the laser and the outputpower of the laser. The output power of the laser is the amount ofenergy the laser outputs per unit time. The fluence of the laser is aproduct of the irradiance of the laser and the exposure time and is thetotal amount of energy delivered by the laser to an area. The amount ofablation depends on the dose: the greater the laser intensity or fluenceon the substrate, the greater the applied dose, and the greater amountof biomolecule ablation. Typically the area receiving the applied doseand which is used to determine the intensity or fluence is defined bythe diameter of the intersection of laser focal volume 14 with thesurface of substrate 10. This area is typically called the laser spot.For example, if the laser is focused onto a glass coverslip, the areaused to determine the dose is that of the laser spot on thecoverslip—that is the intersection of the focal volume at its narrowestwith the surface of the coverslip.

Laser 20 may be focused as to increase intensity and decrease size of alaser focal volume 14 that is applied to substrate 10. A diameter oflaser 20 at laser focal volume 14 that interacts with one or morebiomolecules 12 on substrate 10 may be as small as diffraction limitedand as large as permitted by available power to achieve ablation. Laser20 may deliver the laser radiation to substrate 10 by a near-fieldsource, for example, an optical fiber with an aperture at a front endwith a diameter smaller than a wavelength of light, positioned withinone wavelength of substrate 10 that produces an irradiated area smallerthan a diffraction-limited spot.

Laser 20 may be of any wavelength or combination of wavelengths that canablate function or activity of one or more biomolecules, such aswavelengths between about 190 nm and about 11 μm. Laser 20 can be pulsedor continuous wave. The pulses can be of any length and number that issufficient, given other laser parameters including power and focus, toachieve ablation, such as pulses of about 1×10⁻¹⁵ seconds to about 1×10²seconds.

Laser 20 may be written over a substrate 10 by scanning laser 20, forexample, as shown in FIG. 1D, and/or translating a stage holdingsubstrate 10 in all three axes with respect to an optical axis of laser20. Substrate 10 may be rotated in all three axes with respect to anoptical axis of laser 20. Laser 20 translates a position of the laserradiation on substrate 10. Laser 20 may translate the position of thelaser radiation by shutter 22 and/or a mirror 24.

Writing laser 20 over substrate 10 is a serial process. A computer maycontrol scanning laser 20 and/or translating the stage holding substrate10 and/or a shutter 22 that prevents or allows the application of laser20 to one or more biomolecules 12 on substrate 10. One or moreoperator-defined predetermined patterns and/or laser parameters may beprogrammed into the computer. The computer is described herein by way ofexample as control processing unit. Of course, it is contemplated by thepresent disclosure for the computer to include any programmable circuit,such as, computers, processors, microcontrollers, microcomputers,programmable logic controllers, application specific integratedcircuits, and other programmable circuits. It is further contemplated bythe present disclosure that the computer is any number of controldevices providing various types of control, e.g., centralized,distributed, redundant and/or remote control.

Production of arbitrary patterns on substrate 10, as shown in FIGS. 1Eand 1F, is possible by writing laser 20 over substrate 10. An arbitrarypattern includes of a non-ablated portion 17 that includes one or morebiomolecules 12 of at least one functional and/or active biomolecule andan ablated portion 15 of at least one wholly or partially ablatedbiomolecule distributed in any fashion on substrate 10. Ablated portion15 is any region that can be physically realized by translation orrotation of the laser radiation relative to substrate 10 or translationand rotation of substrate 10 relative to the laser radiation, in one,two, or three dimensions. An arbitrary pattern includes points, lines,polygons, smooth irregular shapes, and regular or irregular volumes.

A range of function and activity of ablated biomolecules within ablatedportion 15 of the arbitrary pattern can be from 100% functional and 100%active to 0% functional and 0% active. The arbitrary pattern may includea complete and whole biomolecule, a fraction of the biomolecule, or acomplete absence of the biomolecule, or any combinations thereof. Thedimensions of features of active or functional biomolecules in thearbitrary pattern are as small as less than about 1 nm and as large as 1meter or as large as permitted by a translation system that changes therelative position of the substrate 10 to that of laser 20. Shapes offeatures of the arbitrary pattern are limited by dimensions of thefocused laser spot on substrate 10 that ablates one or more biomolecules12 that form ablated portion 15. Feature dimensions may be achieved byapproaching a center of a feature of the pattern from two or more sideswith laser radiation, ablating each side until as few as one functionalor one active molecule remains in the feature. The one or more ablatedportions having at least one lateral dimension in the plane of thesubstrate from at least 0.1 nanometer, at least 1 nanometer, at least 10nanometers, at least 100 nanometers, or at least 250 nanometers to 1meter. One or more non-ablated portions is on the substrate. The one ormore non-ablated portions having at least one lateral dimension in theplane of the substrate from about at least 0.1 nanometer, at least 1nanometer, at least 10 nanometers, at least 100 nanometers, or at least250 nanometers to 1 meter (lateral dimension). The ablated portions haveless than 100% of biological function or activity, or biochemicalfunction or activity of the one or more biomolecules on the substrate.

Gradient patterns of one or more biomolecules 12 on substrate 10 may becreated by laser 20. A gradient pattern is an arbitrary pattern in whichan extent of activity or function per unit distance, area, or volume,and any combination thereof varies with position on a substrateuniformly coated substrates. Multiple independent operational parametersdetermine the extent of a gradient, and because writing with laser 20 isa serial process, operational parameters can be varied continuouslyduring writing to generate gradients over length scales from least 0.1nanometer, at least 1 nanometer, at least 10 nanometers, at least 100nanometers, or at least 250 nanometers to 1 meter. Operationalparameters include an optical system, a wavelength of a laser, a pulserate and a pulse duration (if pulsed) of a laser, an output power of alaser, a laser intensity, a laser fluence, an energy density at a thefocus, a scan or translation speed of a substrate relative to a laserfocus, a mode of a laser, a size and beam quality (M²) of a focusedlaser spot.

One or more biomolecules 12 have a portion that is ablated with laser 20in the predetermined pattern. Ablation includes inactivation ofbiomolecules or biological functionality or destruction or removal ofbiomolecules. The predetermined pattern of one or more biomolecules 12includes an ablated portion 15 and a non-ablated portion 17 thatincludes one or more biomolecules 12. Ablated portion 15 includes fullyor partially inactive or non-functional biomolecules or fully orpartially destroyed biomolecules or includes no biomolecules (they havebeen removed) and non-ablated portion 17 includes active and functionalbiomolecules.

Ablated portion 15 and non-ablated portion 17 can be of uniform ornon-uniform height. In contrast to applying biomolecules in a pattern ona substrate, ablating ablated portion 15 of one or more biomolecules 12forms the predetermined pattern by removing or inactivating the portionof one or more biomolecules 12. The predetermined pattern can be eitherpositive, in which non-ablated portion 17 contains one or more ofbiomolecules 12, or negative. A negative predetermined pattern ablatesan ablated portion and backfills the ablated portion with one or morebiomolecules. Any biomolecules may backfill the ablated portionincluding biomolecules that are the same as or different than the one ormore biomolecules in non-ablated portions.

Coating substrate 10 with one or more biomolecules 12 is independent ofablating ablated portion 15 of one or more biomolecules 12 with laser 20in the predetermined pattern, so that the coating and the predeterminedpattern may be formed in a wide range of environments. The environmentfor coating the substrate 10 with a one or more biomolecules 12 and theenvironment for ablating the predetermined pattern with laserirradiation can be different environments. The range of environments forablation may include biomolecules that are dry in an ambient atmosphere;dry in a selected gas that may influence the patterning process;biomolecules that are covered by a thin layer of liquid; biomoleculesthat are hydrated by a humid environment; biomolecules that are hydratedby an aqueous environment; biomolecules that are maintained in anon-aqueous environment; biomolecules that are maintained in a aqueousenvironment; and/or biomolecules that are in a vacuum. Substrate 10 maybe submerged in water for ablating ablated portion 15 of one or morebiomolecules 12 with laser 20. For example, FIG. 5 shows a pattern ofregions of ablated fibronectin 85 and regions of non-ablated fironectin82 on a substrate in water. FIG. 5 includes irregular streaks across thesurface as a result from inadvertent mechanical abrasion subsequent topatterning.

Referring again to FIG. 1, ablation can occur within or in proximity toa focal volume 14 of laser beam 21, and can result from photochemical,photothermal, or photophysical interactions or combinations of the threeclasses of interactions of the laser radiation with a biomolecule, asubstrate, a coupling agent, or combinations of thereof. Photochemicaleffects include electronic excitation and subsequent bond breaking ofspecific chemical bonds within the substrate, the biomolecule, orcoupling agent. Photothermal effects include heating of the biomolecule,substrate, or coupling agent by laser 20, and subsequent thermalresponse to a change in local temperature. For example, a biomoleculemay undergo a change of conformation at increased temperature or atleast one bond within that biomolecule may break. Photophysical effectsinclude interactions, for example in an electrostatic or ballisticmanner, of any removed material with the biomolecules, substrate, orcoupling agent. Photophysical also includes interactions of alaser-generated plasma with the biomolecules, substrate, or couplingagent.

A degree of ablation varies with, wavelength of the laser, opticalproperties of the substrate and biomolecule, absorptive properties ofthe substrate and biomolecule, and thermal properties of the substrateand biomolecule, the species of biomolecule, and the optical,absorptive, and thermal properties of the coupling molecules. Exposuretime of substrate 10 to focal volume 14 of laser beam 21 can range fromthe duration of a single pulse (1 femtosecond at the current state ofthe art) to seconds. Laser intensity can range from 0 Joules/centimeter²(J/cm²) to the maximum provided by the current state of the art. Thepractical upper bound will be no greater than the intensity that candamage substrate 10. For example, for a glass substrate and a PALM laserdescribed herein, the upper bound is approximately 200microJoules/pulse. Laser wavelengths can range from deep UV (<200 nm) tofar infra red (>10 μm). Substrate optical properties can range fromopaque to transparent to a specific laser wavelength. Substrates can bereflective. Laser 20 may have an intensity that forms a microplasma thatcauses ablation upon contact of the microplasma with one or morebiomolecules 12. Laser 20 may have a wavelength that excites one or morebonds within one or more biomolecules 12 and thereby causes ablationtherein. Laser 20 may transfer heat or irradiate one or morebiomolecules 12 or substrate 10, thereby causing ablation.

The predetermined pattern including one or more ablated portions 15 andone or more non-ablated portions 17 may be used for controlling cellshape or function of fibroblasts 116. For example, FIG. 1F shows anillustrative fluorescence micrograph of fibroblasts 116 attached to apatterned fibronectin 112 coated surface including ablated fibronectin115 and functional fibronectin 117 that includes fibronectin 112. Thepredetermined pattern including one or more ablated portions 15 and oneor more non-ablated portions 17 may be used to study or control neuronalguidance; to study or control cellular migration; to study or controlcell division; to study or control and other biological processes; tostudy or control engineered tissue constructs including vascularconstructs; to study or control functional properties of cells includingsecretion of important proteins (e.g. insulin); to study or controlmechanical properties including elastic modulus and adhesive force; tostudy or control intra- and inter-cellular signaling; to make in vitrodiagnostic assays including microarrays; or any combination thereof.

In one example of patterning a Zeiss PALM microbeam laser dissectionmicroscope with a 337.1 nanometer wavelength pulsed laser with pulses ofless than 4 nanoseconds (ns) and of 300 microjoules/pulse at a 30 Hertz(Hz) repetition rate, with a motorized stage, and with the PALM ROBOsoftware was used. A predetermined pattern was drawn using the PALM ROBOsoftware. Parameters such as laser energy, write speed, and UV focuswere selected to optimize the predetermined pattern or portion of apattern. Optimized operating parameters included a UV energy of 60%, acut speed of 10 μm/s, and a UV focus value of 49. The accessible rangefor the UV energy parameter was between 0% and 100%. The accessiblerange for the cut speed was between 0 μm/s and 70 μm/s. The accessiblerange for the UV focus value was between 0 and 100. A 40×, 1.4 NAobjective was used to focus the laser energy onto the substrates. Thelaser energy value is proportional to the intensity of the laser, and itis proportional to the applied does of the laser on the substrate.

FIG. 6A is an immunofluorescence image of a surface coated withsurface-bound fibronectin 92, where fibronectin 92 is stained with anantibody against fibronectin, and a pattern of regions of ablatedfibronectin 95 was formed by point exposures of a CO₂ laser with awavelength of 10.6 μm, and the fluorescence intensity of the fibronectinstaining is proportional to the activity of the fibronectin. Thefibronectin activity in the pattern ranges from high (light areas) tolow (dark areas). FIG. 6B is a graph showing a plot of fluorescenceintensity as a function of position from left to right within the dashedline in FIG. 6A.

FIG. 7A is an immunofluorescence image of a surface coated withsurface-bound fibronectin 102, where fibronectin 102 is stained with anantibody against fibronectin, in a pattern 105 formed by a single pointexposure of a YAG laser with a wavelength of 1064 nanometers, and thefluorescence intensity of the fibronectin staining is proportional tothe activity of the fibronectin. The fibronectin activity in the patternranges from high (light areas) to low (dark areas). FIG. 7B is a graphshowing a plot of fluorescence intensity as a function of position fromleft to right within the dashed line in FIG. 7A.

FIG. 3A illustrates an immunofluorescence image of a surface coated withsurface-bound fibronectin, where the fibronectin is stained with anantibody against fibronectin. A pattern including a series of verticallines 60 was formed by different doses of a laser shows dose dependenceof inactivation of surface-bound fibronectin with the dose increasingfrom left to right. Each line of vertical lines 60 was exposed to adifferent laser intensity with laser intensity increasing from left toright in FIG. 3A. The laser intensity is proportional to the UV energyparameter of the Zeiss PALM laser system.

Functional activity of the fibronectin in exposed regions 62 andunexposed regions 64 was quantified by immunofluorescence andfluorescence microscopy. The fluorescence intensity is presumed to beproportional to fibronectin activity. As shown in FIG. 3B, normalizedfibronectin activity, measured by immunofluorescence resulting from theantibody against fibronectin, varies as a function of dose of thesurface of FIG. 3A; thus, the laser inactivates substrate-attachedprotein in a dose-dependent manner. At low laser intensities, forexample, 50% laser intensity, the plasma inactivates a fraction of thetotal protein activity on the substrate and at greater intensities, forexample, greater than 60% laser intensity, there is greater or totalinactivation of protein activity, as shown in FIG. 3B. Intensitiesgreater than 70%, for example, may damage the coverslip. At sufficientlyhigh intensities, the plasma removes biomolecules from the substrate aswell as inactivates the biomolecules.

Referring to FIGS. 4A through 4C, inactivation can be achieved withoutremoval of mass that is measurable by an atomic force microscope. Asshown in FIG. 4A, an atomic force microscope topographic image of asurface coated with surface-bound fibronectin 72 has a pattern formed byablated fibronectin 75. FIG. 4B shows an immunofluorescence image ofFIG. 4A, where following the topographic imaging the fibronectin wasstained with an antibody against fibronectin. FIG. 4C shows a first plot77 of fluorescence intensity (thick line) as a function of position fromleft to right within a dotted box 76 of FIG. 4B. FIG. 4C shows a secondplot 78 showing height from left to right within dotted box 76 of FIG.4B of the atomic force microscope topograph (thin line).

Referring now to FIG. 2A, the method for ablating biomolecules on asubstrate with a laser to form a predetermined pattern including one ormore ablated portions and one or more non-ablated portions may include atube 50 having a solvent 54 within a tube interior. Tube 50 istranslucent, such as, for example, a glass capillary. The method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions may include coating one or more biomolecules12 on an interior surface of a tube wall of tube 50 in solvent 54, asshown in FIG. 2B. Laser 20 irradiates through the tube wall to interiorsurface having one or more biomolecules 12 coated thereon, as shown inFIG. 2C. As shown by arrows 56 and 58 in FIGS. 2D and 2E, translatinglaser beam 21 with mirror 24, activating and deactivating laser beam 21by shutter 26, rotating or translating tube 50, or any combinationsthereof exposes areas of one or more of molecules 12 to laser beam 21.The radiation of laser beam 21 ablates one or more one or morebiomolecules 12 forming one or more ablated portions 15 in apredetermined pattern, as shown in FIG. 2D. FIG. 2E shows cultureadherent cells 57 on the interior surface of tube 50. Growth andfunction of culture adherent cells 57 are influenced by ablated portions15 of the predetermined pattern of the one or more biomolecules 12.

The predetermined pattern on tube 50 can be use for a wide range ofapplications, including protein/nucleic acid biochip sensors, cell-basedsensors, lab-on-a-chip assays, lab-in-a-capillary assays, cell adhesionassays, cell translocation/migration/invasion/chemotaxis assays,neuronal-guidance assays, tissue-engineering, biomedical devicebiocompatability, vasculature regeneration, control of cellularbiological processes such as adhesion, migration, and division, controlover functional properties of cells such as secretion, shape mechanics,and intra and intercellular signaling.

To establish laser operating parameters for using the method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions for patterning any particular biomolecule, thelaser parameters that influence the ablation process can be examined.The amount of laser radiation delivered to the biomolecules on thesurface can be described by the applied dose of the laser. There are twocomponents to the dose of the laser—the intensity of the laser on thebiomolecules and the total time the biomolecules are exposed to thelaser. The intensity, also called power density and irradiance, of thelaser is the amount of energy per unit time per unit area the laserdelivers to the substrate. The intensity depends on the optical systemof the laser and the output power of the laser. The output power of thelaser is the amount of energy the laser outputs per unit time. Thefluence of the laser is a product of the irradiance of the laser and theexposure time and is the total amount of energy delivered by the laserto an area.

Parameters for a continuous wave (CW) laser that contribute to theapplied dose of a CW laser can include the laser wavelength, the laseroutput power, the exposure time, the spatial mode, and the size andquality of the focused laser spot on the substrate. The wavelength ofthe laser can be selected based on the known physical chemistry of thebiomolecules being ablated, or wavelengths can be tested exhaustively byincrementing the wavelength 10 nm at a time from about 190 nm to 11 μm.For each wavelength range of intensities and doses can be examined. Theintensities, for example, can be varied from 0 W/μm² to 5 W/μm² byadjusting the output power of the laser, and the exposure times canrange from 1 μs to 1 s, resulting in a fluence that varies from 0 J/μm²to 5 J/μm². Each combination of parameters can be used to expose an areaon substrate 10 coated with the biomolecule of interest, where this areais large enough for suitable analysis, for example by atomic forcemicroscopy or immunofluorescence microscopy. The substrate then can beanalyzed to determine the function or activity of the biomolecule orbiomolecules in the irradiated area. The activity or function can bedetermined by for example enzymatic activity, fluorescence, antibodybinding, ligand binding, cell binding, cell repulsion or any suitablefunctional assay for the biomolecule in question. Once the appropriatecombination of parameters for ablation is established, these parameterscan be used in method for ablating biomolecules on a substrate with alaser to form a predetermined pattern including one or more ablatedportions and one or more non-ablated portions to pattern the biomoleculeof interest.

Parameters for a pulsed laser that contribute to the applied dose of apulsed laser can include the laser wavelength, the laser pulse width,the laser pulse frequency, the laser output peak power, the exposuretime, the spatial mode, and the size and quality of the focused laserspot on the substrate. The wavelength of the laser can be selected basedon the known physical chemistry of the biomolecules being ablated, orwavelengths can be tested exhaustively by incrementing the wavelength 10nm at a time from about 190 nm to 11 μm. For each wavelength range ofintensities and doses can be examined. Pulsed lasers in general havegreater intensities than CW lasers due to the short pulse times(typically on the order of 10⁻⁹ s, 10⁻¹² s, or 10⁻¹⁵ s for the currentstate of the art). The intensities, for example, can be varied from 0W/μm² to 100 kW/μm² by adjusting the output power of the laser, and theexposure times can range from 1 μs to 1 s. Each combination ofparameters can be used to expose an area on a substrate coated with thebiomolecule of interest, where this area is large enough for suitableanalysis for example by atomic force microscopy or immunofluorescencemicroscopy. The substrate then can be analyzed to determine the functionor activity of the biomolecule or biomolecules in the irradiated area.The activity or function can be determined, by for example enzymaticactivity, fluorescence, antibody binding, ligand binding, cell binding,cell repulsion or any suitable functional assay for the biomolecule inquestion. Once the appropriate combination of parameters for ablation isestablished, these parameters can be used in method for ablatingbiomolecules on a substrate with a laser to form a predetermined patternincluding one or more ablated portions and one or more non-ablatedportions to pattern the biomolecule of interest.

Many different types of lasers can be used for the method for ablatingbiomolecules on a substrate with a laser to form a predetermined patternincluding one or more ablated portions and one or more non-ablatedportions. To determine if a laser can be used, a test of relevantparameters can be carried out. For example, a pulsed 10 Watt YAG laserwith a 50 kHz pulse frequency is tested by irradiating a substrate 10coated with biomolecules 12. The laser is focused 14 onto thebiomolecules. The substrate is exposed to the laser on points atdifferent positions on the substrate for varying lengths of time, forexample 1 μs, 10 μs, 100 μs, 1 ms, 10 ms, 100 ms and 1 s at a series ofdifferent output powers, for example, 0.5 W, 1 W, 5 W, ad 10 W. Thesubstrate is then analyzed to determine the function or activity of thebiomolecule or biomolecules in the irradiated area. The activity orfunction can be determined by for example enzymatic activity,fluorescence, antibody binding, ligand binding, cell binding, cellrepulsion or any suitable functional assay for the biomolecule inquestion. Once the appropriate combination of parameters is established,these parameters can be used in method for ablating biomolecules on asubstrate with a laser to form a predetermined pattern including one ormore ablated portions and one or more non-ablated portions to patternthe biomolecule of interest. A similar approach can be used to determinethe optimal parameters for a continuous wave laser system. The selectionof lasers may also be guided by physical chemical properties of thebiomolecules being patterned, by someone skilled in the art.

EXAMPLE 1 Cellular Migration

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control cellularmigration. The secreted protein vascular endothelial growth factor(VEG-F) induces cell migration in vitro. Most experiments on this kindare performed using one or more point sources of diffusible VEG-F, andthe migration of cells towards that source is observed over time.Although this system has produced substantial information about thebiology and biochemistry of cell migration, this source-and-diffusionmodel of VEG-F is restricted to distributions of molecules that areachieved with combinations of sources of diffusible VEG-F, and it isexceedingly difficult to characterize in terms of the concentrationgradients and the change of the gradients over time. The effects ofcomplex gradients of VEG-F activity on endothelial cell function arestudied using method for ablating biomolecules on a substrate with alaser to form a predetermined pattern including one or more ablatedportions and one or more non-ablated portions in an aqueous environment.A bi-functional polyethyleneglycol (PEG) with one end functionalizedwith a silanol group and another end functionalized with an NHS-esterare used to coat a glass surface with VEG-F. The silanol moiety willbind covalently to the glass surface, and the NHS-ester will bind toamines in the VEG-F molecule. The PEG linker region ensures that theVEG-F is sufficiently free to diffuse in a small (typically 5-100 nmradius), but tethered volume in the aqueous solution. The gradient istime invariant at dimensions larger than the tether distance and arecharacterized, for example by immunofluorescence microscopy. Thegradients produced are spatial distributions of VEG-F that are otherwiseaphysical and that are not possible to produce by any physicallyrealizable combination of sources of diffusible of VEG-F. A surfacecoated with VEG-F as described and patterned in solution using methodfor ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are used to induce endothelial cells tomigrate towards the regions on the substrate that have greater number ofactive VEG-F proteins. Complex patterns of active and inactive VEG-F areused to direct the migration of endothelial cells to specific regions onthe substrate.

EXAMPLE 2 Gradient Pattern of a Growth Factor

To make a gradient pattern of a biomolecule on a substrate, the applieddose of the laser must be modulated as a function of position within thepattern. Modulating the dose can include maintaining a constant powerdensity and varying the speed at which the substrate translates relativeto the laser focus, maintaining a constant speed at which the substratetranslates relative to the laser focus and varying the laser powerdensity as a function of position.

A glass coverslip substrate is cleaned and coated with APTES. Abiomolecule, such as the normally soluble growth factor VEGF, isattached to the coverslip using a tethering molecule such as abifunctional polyethylene-glycol (PEG). The laser system is programmedto make a gradient pattern in which a rectangular area, for example, ofthe VEGF coated substrate is exposed to laser irradiation such thatalong one axis of the rectangle the applied dose of the laserirradiation is increased from a minimum to a maximum. This isaccomplished by using a fixed laser intensity (532 nm wavelength, 0.2 Wlaser output focused with a 60×, 1.4 NA objective) and varying the speedat which the sample translates relative to the laser focus. The longerthe focus remains on a single spot, the larger the applied dose of laserirradiation and the greater the ablation of the biomolecules exposed.Varying the speed from 0.001 μm/s to 100 μm/s will vary the applied doseby 5 orders of magnitude, and vary the ablation from a minimum (at 100μm/s) to a maximum (at 0.00 μm/s). It is possible that the allbiomolecules will be ablated at a dose below the maximum, and that thereis a dose which ablates all biomolecules.

A second method to vary the dose it to keep constant the translationspeed but vary the laser intensity as a function of laser focus positionon the substrate.

EXAMPLE 3 Ablation in Solution

In this implementation, a glass coverslip is coated with fibronectin. Anaqueous solution, PBS for example, is added to the coated surface of thecoverslip, and the coverslip is placed on the stage of the laser system.The laser is focused to the surface of the coverslip coated with thefibronectin and that is in contact with the solution. The laser systemparameters are set to inactivate the fibronectin, but not to remove itfrom the substrate and not to damage the coverslip (for example in thePALM system, laser power set to 55%, scan speed of 25 μm/sec, and a UVfocus of 49), in a predefined pattern.

EXAMPLE 4 Neuronal Guidance

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control neuronal guidance.Biomolecules that guide neuronal cells during development, regeneration,wound healing and other processes include molecules from the molecularfamilies of ephrins, semaphorins, slits, and netrins, and specificneuronal guidance molecules including brain-derived neurotrophic factor(BDNF) and neuronal growth factor (NGF). Neuronal growth guidance isattractive or inhibitory. For example, the semaphorins are a class ofsecreted and membrane bound molecules that guide the axonal growth cone.They are short-range inhibitory signaling molecules. For example, onedifficulty with studying neuronal guidance in vitro withcell-membrane-bound semaphorins is that the spatial distribution ofsemaphorins on a layer of cells in a Petri dish, for example, isdifficult to control. Method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions are used tocontrol the spatial distribution of active and inactive semaphorinsfound in the cell membrane. A substrate that is coated with a specificsemaphorin molecule from a cell membrane or the active binding domain ofthat molecule, and that is patterned using the method for ablatingbiomolecules on a substrate with a laser to form a predetermined patternincluding one or more ablated portions and one or more non-ablatedportions have regions of inactivated semaphorin molecules, regions ofactive semaphorin molecules, and if gradient patterning is used, regionscomposed of combinations of inactive and active semaphorins. Neuronsplated and grown on such a substrate will move in a way that isinfluenced by the regions containing active semaphoring molecules andthe regions containing inactive semaphoring molecules.

EXAMPLE 5 Cell Division

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control cell division. Ithas been shown using micro-contact printed patterns of extra cellularmatrix proteins that the ECM geometry defines the axis of cell polarity(Thery et al., 2006) and guide the orientation of the cell division axis(Thery et al., 2005). ECM proteins such as fibronectin are attached to asubstrate and patterned using method for ablating biomolecules on asubstrate with a laser to form a predetermined pattern including one ormore ablated portions and one or more non-ablated portions. Regions ofactive fibronectin that support the adhesion of a single or multiplecells are contemplated. The effect of the geometry of the ECM pattern onaspects of cell division such as the expression of specific genes, thetiming of key events, the internal organization of organelles and otherintracellular structures in the mother and daughter cells, the axis ofcell division, the properties and dynamics of DNA replication, and theother phenomena that occur during mitosis, are examined and studiedusing standard cell biological techniques such as optical andfluorescent microscopy during each phase of mitosis. Processes relatedto cell division are also controlled. A HeLa cell, for example, platedon a 20 μm×60 μm rectangle of active fibronectin molecules surrounded byan expanse of inactive fibronectin has a high probability that itsdivision axis will be parallel to the width of the rectangle. Theeffects of confinement to the rectangle on internal cellular structuressuch as the cytoskeleton are examined using optical or fluorescentmicroscopy while the cell is on the pattern. Other shapes and geometriesare made as well with method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions for this purpose.Another embodiment of this example is to use substrates with a set ofpatterns, and then examine the combined effect of another factor orfactors, such as soluble molecule or an environmental insult like atemperature change, on cell division.

EXAMPLE 6 Biological Processes

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control and otherbiological processes. The expression of genes within a cell is regulatedby the interactions of a cell with a pattern of ECM proteins on asubstrate (Chen et al., 1997; Dike et al., 1999). Patterns of ECMproteins, signaling molecules, growth factors, growth inhibitors, andother biomolecules are made using method for ablating biomolecules on asubstrate with a laser to form a predetermined pattern including one ormore ablated portions and one or more non-ablated portions to study orcontrol biological processes such as gene expression, differentiation,metabolism, syntheses of proteins, degradation of proteins, secretion,ingestion, growth, apoptosis, spreading, rounding, inter andintracellular communication and signaling, regulation of cellularfunction, and others. For example, to study the effect of a pattern ofepidermal growth factor (EGF) on DNA synthesis within Swiss 3T3fibroblasts using the method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions, a surface isfirst be coated with EGF using a bifunctional PEG linker molecule asdescribe above. Then predefined patterns of inactive EGF and active EGFare made according to method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions, and thefibroblasts plated on the substrates and allowed to adhere, spread, andgrow on the patterns. Assays to detect DNA synthesis (for example thepercentage of nuclei labeled with 5-bromodeoxyuridine after 24 hours)are then be applied to the cells.

EXAMPLE 7 Engineered Tissue Constructs

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study, control engineered tissueconstructs such as vascular constructs. Angiogenesis, the growth ofblood capillaries, is regulated by soluble growth factors and insolubleextracellular matrix (ECM) molecules (Ingber, 1992). Vascularendothelial cells grown to confluence on a substrate with a uniformcoating of ECM molecules do not have all the properties of a vasculaturein vivo. A substrate patterned with ECM proteins using method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions in such a way as to introduce into onto thesubstrate a directional bias provide a more natural environment for theendothelial cells to grow, and thus result in a better model of vasculartissue for study or a better engineered substrate for large-scaleproduction of engineered tissue. For example, parallel lines 5 μm wideof inactive fibronectin on a substrate of active fibronectin moleculescause the plated cells to orient themselves in the direction of thelines. If the cells are grown to confluence on such a pattern, eventhough the cells may grow over the inactive fibronectin, thetwo-dimensional tissue will have different mechanical properties in thedirection parallel to the lines versus in the direction perpendicular tothe lines, and will have mechanical properties that are morewell-defined than those of layers of cells grown without control.

EXAMPLE 8 Functional Properties of Cells

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control functionalproperties of cells such as secretion of important compounds (e.g.insulin, triglycerides, steroids) or other, unnatural, non-native, ornovel behaviors or functions of cells. It is known that the size ofB-cells contributes to the insulin secretion, and that the environmentand shape of mammary epithelial cells in vitro contributes to thesecretion of triglycerides of milk fat. Substrates of ECM molecules suchas fibronectin are patterned using method for ablating biomolecules on asubstrate with a laser to form a predetermined pattern including one ormore ablated portions and one or more non-ablated portions to identifyand patterns of ECM protein that increase the secretion of these andother compounds from appropriate cell types (e.g. B-cells, epithelialcells, granulosa cells). To identify patterns of ECM proteins thatincrease secretion of insulin from B-cells, for example, a series ofpatterns are made using method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions thatsystematically vary in area, perimeter, geometry, and ECM protein.B-cells are plated and grown on substrates containing the patterns, andthe levels of insulin secretion are measured. That pattern are optimizedby examining a insulin secretion for a large number of highlydistinctive shapes in the pattern, taking the shapes that produce thebest insulin expression and varying the shapes in a narrower range ofdimensions, and repeating this process until a pattern that promoteshigh levels of insulin express is identified. A pattern that isoptimized for insulin secretion can then be determined and used fortherapeutic, research, or diagnostic purposes.

EXAMPLE 9 Mechanical Properties of Cells

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are used to study or control mechanicalproperties such as elastic modulus and adhesive force. It has been shownfor ECM proteins patterned on substrates that the pattern geometryaffects cytoskeletal protein organization (Berg et al., 2004) throughinteractions between the ECM proteins, the integrins, the focal adhesioncomplex, and other intercellular protein complexes. The cytoskeletalorganization is the major contributor to cellular mechanics, which isquantified by, for example, the elastic modulus and the Poisson ratio.The mechanical properties of cells are important to their function. Theadhesive force, for example, is a measure of the strength of adhesionbetween a cell and the substrate. Cells with a low adhesive force areeasily removed from the surface under exposure to an applied flow, forexample, but cells with a high adhesive force are not. Substrates withECM proteins, for example fibronectin, patterned using method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions to include regions of active and inactivefibronectin of specific geometries that are used to modulate thecytoskeletal organization of cells plated on those patterns, and thusmodulate the mechanical properties of the cells. Such patterns used inconjunction with assays for the mechanical properties of cells and/orthe intercellular and intracellular signaling pathways that regulate themechanical properties of cells elucidate the mechanisms by which theenvironment controls cellular mechanics. Such patterns can be used tocontrol cellular mechanics for purposes such as tissue engineering,assay development, and biomedical research.

EXAMPLE 10 Intra- and Inter-Cellular Signaling

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to study or control intra- andinter-cellular signaling. Controlling various cell functions andproperties through the use of substrates patterned using method forablating biomolecules on a substrate with a laser form a predeterminedpattern including one or more ablated portions and one or morenon-ablated portions. In order for the cells to respond to the patterns,there must be a signal delivered from the cell surface to the interiorof the cell, often to the nucleus and the proteins and biomolecularcomplexes that regulate gene transcription. Thus in combination withassays to identify and characterize specific components of a signalingpathway or cascade within a cell or a collection of cells, patterns ofbiomolecules such as ECM proteins, growth factors, growth inhibitors,and cytokines, the pathways that cells use to react to their environmentare investigated. For example, to determine the effect of patterns ofepidermal growth factor (EGF) on the activity of the S6 kinase, patternsof active and inactive EGF on substrates are made using method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions. Cells plated on those patterns bind to thesubstrate attached EGF through EGF-receptor proteins on the cellsurface. This binding induces a signal cascade that eventually includesactivation of the S6 kinase. Assays for the S6 kinase activity areapplied to these cells and the influence of EGF pattern shape on S6kinase activity is measured. Cells in contact with other cells, such asthose in a tissue or those grown to confluence in vitro, signal to eachother by secretion of signaling molecules, direct contacts like gapjunction channels, and mechanical linkages through stress fibers. Theeffect of the spatial distribution of biomolecules on the substrates ofthese collections of cells are examined and exploited using method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions. For example, method for ablating biomoleculeson a substrate with a laser to form a predetermined pattern includingone or more ablated portions and one or more non-ablated portions isused to make large islands of ECM protein like fibronectin onto which anumber of cells are plated and grown to confluence on that island.Assays for inter and intra cellular signaling are applied to cells onthe islands to determine the effect of, for example, island size, numberof neighbors, mechanical properties of the collection of cells, on cellsignaling pathways.

EXAMPLE 11 In Vitro Diagnostic Assays

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions is used to make in vitro diagnostic assays,including microarrays or cell arrays. In vitro diagnostic assays, invitro high throughput, and in vitro high content screening assays,including those based on cellular responses to drugs, toxins, pathogens,therapeutic agents, biological and biochemical compounds, and the like,often require the identification and analysis of cells grown in vitro.Typically, such cells are randomly positioned over the substrate, andidentifying and analyzing cells is a time consuming and expensiveactivity. Human expertise or automated image processing and analysisalgorithms are used to identify and analyze cells in micrographs of thesubstrates. Patterns of ECM proteins such as fibronectin that include ashape defined geometry, for example a solid circle, 50 μm in diameter,of fibronectin, that is repeated many times over an area of a substratesuch that each circle was in a predetermined location, for example arectangular array of 100 μm pitch in each direction of solid fibronectincircles, reduce the time required to identify and analyze cells in theassay. The collection of micrographs, the identification of cells, andthe analysis of the cellular response to the analyte(s) are fasterbecause the cells are positioned in pre-defined locations. The repeatedshapes are further optimized for the particular response. For example,an assay that screens for compounds that affect the expression of aparticular gene in a specific cell line are based on repeated patternsof ECM protein that have been identified to optimize the expression ofthat gene in cells plated on those patterns. Compounds that inhibit orenhance the gene expression are then applied at different concentrationsto substrates with cells plated on the patterns, the gene expressionmeasured and correlated to the compound and the concentration.

EXAMPLE 12 Interior Surfaces

Another application of method for ablating biomolecules on a substratewith a laser to form a predetermined pattern including one or moreablated portions and one or more non-ablated portions include one ormore of biomolecules 12 (FIG. 1) coated on an interior surface of aglass capillary or a translucent tube of a biopolymer, applying laser 20to an exterior surface opposite the interior surface of the glasscapillary, and ablating a one or more of biomolecules 12 in apredetermined patterned on the interior surface of the glass capillary.The predetermined pattern is used for a wide range of applications,including microfluidic devices, protein/nucleic acid biochip sensors,cell-based sensors, lab-on-a-chip assays, lab-in-a-capillary assays,cell adhesion assays, cell translocation/migration/invasion/chemotaxisassays, neuronal-guidance assays, tissue-engineering, biomedical devicebiocompatability, vasculature regeneration, control of cellularbiological processes such as adhesion, migration, and division, controlover functional properties of cells such as secretion, shape mechanics,and intra and intercellular signaling.

EXAMPLE 13 Stem Cell Differentiation

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are applied to substrates to study and controlstem cell differentiation. Patterns of biomolecules on substratescontrol the differentiation of stem cells grown on the patterns. Forexample, adult rat hippocampal progenitor cells (AHPCs) plated onmicropatterns of laminin preferentially acquired neuronal morphologycompared to those AHPCs plated on unpatterned substrates (Recknor etal., 2006). The method for ablating biomolecules on a substrate with alaser to form a predetermined pattern including one or more ablatedportions and one or more non-ablated portions is used, for example, todetermine patterns of biomolecules that direct stem cells todifferentiate into specific cell lines. For example, a set of patternsof active and inactive ECM molecules is made using method for ablatingbiomolecules on a substrate with a laser to form a predetermined patternincluding one or more ablated portions and one or more non-ablatedportions on a set of substrates such that each substrate has the sameset of patterns but is coated with a different type biomolecule. Stemcells, human mesenchymal stem cells (hMSCs) for example, are plated andgrown on each substrate in the set and monitored for differentiation todetermine which ECM biomolecule and which pattern is most efficient fordirecting differentiation to a specific cell type, neurons for example.Another set of patterns are developed based on the results of the firstpattern and biomolecule screen. For example, the dimensions of aparticular class of shapes are varied systematically. hMSCs are platedon the new patterns of selected ECM biomolecules and monitored fordifferentiation. Repeated applications of this approach further refinesthe patterns to more efficiently and reliably direct stem celldifferentiation.

EXAMPLE 14 Information

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are used to study and control the amount ofinformation in a pattern of biomolecules on a surface. The amount ofinformation in a pattern is quantified using information theory(Shannon, 1948; Bell, 1948). Thus, the information in the spatialdistribution of biomolecules patterned on a substrate using method forablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are quantified, and such patterns areclassified according to their information content. The response of cellsplated on patterns with systematically varied amounts of information isexamined and correlated to the information content of the patterns. Forexample, the morphology of cells varies with the information content ofa pattern. Other cellular responses include gene expression, mitosis,differentiation, migration, secretion, apoptosis, inter- andintra-cellular signaling, metabolic responses, and mechanical responses.

EXAMPLE 15 Device Enhancement

Method for ablating biomolecules on a substrate with a laser to form apredetermined pattern including one or more ablated portions and one ormore non-ablated portions are applied to enhance or to modify for newuses, including those described above, pre-existing devices includingmicroscope coverslips, microscope slides, Petri dishes, cell cultureflasks, multi-well plates, test tubes, eppendorf tubes, glass or plasticcapillaries, sensor elements, biomedical implants, and gels.

EXAMPLE 16 Backfilling

A substrate, for example a glass coverslip, is plasma-cleaned for 5minutes in a plasma cleaner (Harrick Plasma; Ithaca, N.Y.), thenincubated for 20 minutes in piranha solution (H₂O₂:H₂SO₄::1:3). Thesubstrate is then rinsed thoroughly in water and dried under nitrogen.Then, the glass coverslip is coated first with APTES. Bovine SerumAlbumin (BSA; Sigma, St. Louis, Mo.) is then coated on top of the APTESlayer by incubating the coverslip in a 1% solution (w/v in phosphatebuffered saline, PBS) for 1 hour. After incubation with BSA solution,the coverslip is rinsed with water and dried under nitrogen. A lasersystem with a parameter set chosen to remove the BSA from the substrate(for example, the PALM system with laser power set to 60%, and scanspeed of 20 μm/s, UV focus at 49, with a 40×0.6 NA objective) is used toablate the BSA in a predefined pattern. Then, the ablated region is thenbackfilled with a biomolecule of interest. To backfill with laminin, forexample, the substrate is incubated in a 0.1 mg/mL solution of lamininin PBS for 2 hours at 37 C. The substrate is then rinsed with water anddried under nitrogen.

The components of the present disclosure are described herein in termsof functional block components, flow charts and various processingsteps. As such, it should be appreciated that such functional blocks arerealized by any number of hardware and/or software components configuredto perform the specified functions. For example, the present disclosureemploys various integrated circuit components, e.g., memory elements,processing elements, logic elements, look-up tables, and the like, whichmay carry out a variety of functions under the control of one or moremicroprocessors or other control devices.

Similarly, the software elements of the present disclosure may beimplemented with any programming or scripting language such as C, SQL,C++, Java, COBOL, assembler, PERL, or the like, with the variousalgorithms being implemented with any combination of data structures,objects, processes, routines or other programming elements. Further, itshould be noted that the present disclosure may employ any number ofconventional techniques for data transmission, signaling, dataprocessing, network control, and the like as well as those yet to beconceived.

EXAMPLE 17 Patterning on the Inside of a Capillary

The laser-based approach allows for patterning on the surfacethree-dimensional structures when there is an optical path available,and it allows for patterning the inside (or side away from the source ofthe laser) of optically translucent materials. To demonstrate the latterpatterned proteins were demonstrated on the inside of a quartz capillary(FIG. 8). The capillary was first coated on the inside with humanfibronectin protein, and air-dried. The capillary was then mounted onthe computer controlled x,y,z stage, and patterning is performed using afrequency multiplied YAG laser that is focused onto the internal surfaceof the capillary. Following patterning the capillary is stained with anantibody against fibronectin, and examined by immunofluorescencemicroscopy. Only the inside of the capillary is treated withfibronectin, and only the inside is stained. Fluorescence microscopicimaging of the pattern was only possible by focusing the microscopethrough the external surface of the capillary, through the walls, andonto the interior surface. Thus the pattern must be on the inside. Thepattern is well-defined and extends from the center of the capillary tothe edges on the top surface.

Other examples of interior surfaces that may be patterned include theinside surfaces of hoses and tubes, flasks, sealed chambers,microfluidic systems, lab-on-a-chip devices, and pipettes. Interiorsurfaces patterned in such a manner may be used for modelingvasculature, capillaries and vessels of the lymphatic and circulatorysystems, and other duct-like biological structures, or as substrates fortissue engineering. They may also be used in fluidic, microfluidic orlab-on-a-chip systems to capture select cells out of a solution(including human serum or whole blood) containing a multitude of cellsflowing past the patterns.

EXAMPLE 18 Patterned Surfaces with Topographic Changes Undetectable byAtomic Force Microscopy Height Imaging

The inactivation-based approach can produce patterns of active andinactive protein without introducing topographic changes detectable byatomic force microscopy (AFM) height imaging. A substrate coated withthe protein fibronectin was patterned using the laser to inactivatelined of protein. AFM was used to characterize the fibronectin-patternedsubstrates, and the height images were compared to immunofluorescencemicroscopy images of the patterned area. Topographic AFM images withsub-nm resolution in z show that there is no detectable difference inthe topography of the surface of the inactivated area and thesurrounding area (FIG. 9, bottom). Following AFM imaging,immunofluorescence microscopy was performed to confirm that the areaimaged by AFM was the patterned area (FIG. 9, upper). Thus the exposureto the laser inactivates the protein without introducing topography intothe sample. Although the exact nature of the inactivated protein is notknown, this result is extremely useful, because it means thattopographic and chemical cues to cells can be uncoupled using thismethod.

EXAMPLE 19 Protein Patterning on a Variety of Materials

The laser-based patterning method can be applied to a wide range ofsubstrate materials used for cell-culture. Glass, silicon, quartz,polystyrene (tissue culture plastic), and polydimethylsiloxane (PDMS)substrates were coated with fibronectin (FN) and inactivated the proteinin simple patterns on the substrates (FIG. 10). These results show thatthe laser-based inactivation approach is flexible with respect tosubstrate material, and thus patterning of the range of existing commonsubstrate materials is possible.

Cell culture substrates can be patterned using this method includesubstrates such as polystyrene Petri dishes, multiwell plates, flasks,microtiter dishes, slides, coverslips, capillaries, pipettes, andchambers; glass-bottomed Petri dishes, multiwell plates, microtiterdishes, flasks, and chambers; glass slides, coverslips, capillaries,pipettes, chambers, Petri dishes, multiwell plates, flasks; PDMS-coatedslides, coverslips, multiwell plates, flasks, capillaries, pipettes, andchambers; and substrates made of PDMS and other elastomeric compounds.

In addition, materials such as glass, silicon, PDMS, and polystyrene canbe used in combination to build complex devices such as microfluidicsystems, biosensors, lab-on-a-chip devices, and implantable devices, andthus components of these complex devices containing such materials canbe patterned with active and inactive protein and assembled into complexdevices.

EXAMPLE 20 Protein Patterning of Common Extracellular Matrix Proteins

Laser-based inactivation patterning of proteins can be used to makepatterns of common extracellular matrix (ECM) proteins. Laser-basedinactivation process was demonstrated to inactivate, in addition tofibronectin (FN), other species of surface-attached ECM proteins.Collagen- and laminin-coated glass and quartz substrates were patternedby the laser-inactivation method, and the resulting patterns weredetected using immunofluorescnce microscopy (FIG. 11). These results incombination with those above show that the method is general and that itcan be applied to different proteins on a variety of surfaces.

Patterns of active and inactive ECM protein can be used to control cellfunction, cell behavior, cell structure, cell shape, cell mechanics,cell fate, cell metabolism, cell division, cell differentiation, cellsignaling, inter- and intra-cellular communication, cell secretion, cellmigration, cell dynamics, cell adhesion and cell spreading.

EXAMPLE 21 Focal Adhesions can Form on Micropatterns of Fibronectin

Patterns of active and inactive fibronectin protein made by thelaser-based method support the formation of focal adhesions. Patterns of5 μm×5 μm fibronectin squares were made and Swiss-3T3 fibroblasts wereplated on them. After the cells spread, they were fixed and stained forfocal adhesions using an antibody against vinculin, a protein componentof the focal adhesion complex. Focal adhesions were detected (FIG. 12),and the focal adhesions appear to form preferentially on the activefibronectin. Similar results were obtained for vascular smooth musclecells. These results further demonstrate that the patterned proteinremains functional on the surface, and that the locations of focaladhesions can be controlled by protein patterns.

EXAMPLE 22 Gradient Patterns of Active and Inactive Proteins

The laser-based inactivation approach can make a variety of gradientpatterns on fibronectin-coated substrates. By varying the exposureparameters as a function of position, linear gradient (FIG. 13A) andlogarithmic gradient (FIG. 13B) patterns of inactivated protein can beproduced. The power of the approach described herein is demonstrated bymaking curved bidirectional gradients (FIG. 13C)—which would be nearlyimpossible by a microfluidic approach, for example. The patterns wereimaged by immunofluoresence microscopy. From the results it is clearthat the method can make defined gradients of defined shape.

The ability to pattern gradients of active and inactive proteins can beused to direct cell migration, cell adhesion and cell spreading, as wellas the self assembly of cells into larger scale structures.

EXAMPLE 23 Large Area Patterning and High Speed Patterning

The laser-based method can be used to pattern large areas of active andinactive protein. Patterning of fibronectin was performed on 1 inchround glass coverslips placed on an 18 inch×24 inch stage that is partof a laser patterning system. The system is designed such that afocusing optical assembly is translated on a moving beam over the entirestage area at linear displacement rates of >10 inches per second (in oneaxis). Objects up to the full lateral dimensions of the stage can beirradiated by a laser that is focused onto the object via the focusingassembly. The position of the laser focus is computer controlled, andcan be moved in any programmable set of motions. This system was used topattern proteins on coverslips placed on different places, spread overthe stage surface (FIG. 14). The coverslips were all patterned withoutmoving them, or without stopping the laser movement. Areas between thecoverslips were not exposed to the laser by turning the laser off as itmoved over those areas. Patterning of more than 50% of the surface of acoverslip (˜2 cm²) with a line or grid pattern, where at least 25% ofthe area within the pattern is inactivated, was accomplished in lessthan 2 minutes (per coverslip). After exposure the coverslips werestained with a polyclonal antibody to fibronectin and visualized byfluorescence microscopy (FIG. 14). Examination of the fluorescencedistribution shows well formed patterns on all the coverslips, and thatthe patterns agree well with the input patterns. These resultsdemonstrate fast patterning of proteins over large areas, with linearprotein ablation speeds of over 10 inches per second, and areal ablationof >10 mm² per second. In this demonstration the coverslips are forpractical purposes not physically connected, but the patterning ofseveral coverslips at the same time demonstrates that patterning overlarge areas—up to the dimensions of the stage it possible. Patterning ofsubstrates larger than the dimensions of the stage is also possible ifthe substrate is moved across the stage—by for example a conveyer belt.

EXAMPLE 24 Creating Gradient Patterns Using the Gabor Expansion

The step sizes and laser powers for making protein gradients may bedetermined by application of the Gabor expansion (Gabor, 1946;Bastiaans, 1980; Janssen, 1981) to the desired pattern function. TheGabor expansion of a function f(x) decomposes it into a sum of Gaussianbasis functions. It can be used in conjunction with the laser-basedpatterning method described herein, because the laser ablation profilecan be Gaussian. The Gabor expansion may be applied to multidimensionalfunctions to create multidimensional gradient patterns. Here theone-dimensional case is described as an example. Gabor expanded afunction f(x) in terms of a basis function g(x) and coefficients a_(mn):

$\begin{matrix}{{f(x)} = {\sum\limits_{m = {- \infty}}^{\infty}\;{\sum\limits_{n = {- \infty}}^{\infty}\;{a_{mn}{g( {x - {m\;\Delta\; x}} )}{\mathbb{e}}^{2\;\pi\;{\mathbb{i}}\; n\;{x/\Delta}\; x}}}}} & (1)\end{matrix}$where Δx is the spacing in variable x, and g(x)=e^((−x) ² ^(/2σ) ² ⁾.In cases where σ≦Δx and Δx is less than the smallest feature in f(x)(i.e.: Δx<π/Q_(B) where Q_(B) is the bandwidth limit of f(x)), theexpansion can be estimated (de Wolf, 1989) as

$\begin{matrix}{{{f(x)} = {\sum\limits_{m = {- \infty}}^{\infty}\;{A_{m}{g( {x - {m\;\Delta\; x}} )}}}}{where}} & (2) \\{{A_{m} = {( {2\;\pi} )^{{- 1}/2}\frac{\Delta\; x}{\sigma}( {{f( {m\;\Delta\; x} )} - {\frac{\sigma^{2}}{2}{f^{''}( {m\;\Delta\; x} )}} + {\frac{\sigma^{4}}{8}{f^{(4)}( {m\;\Delta\; x} )}} + \ldots} )}}{thus}} & (3) \\{{f(x)} \approx {( {2\;\pi} )^{\frac{1}{2}}\frac{\Delta\; x}{\sigma}{\sum\limits_{m = {- \infty}}^{\infty}\;{{f( {m\;\Delta\; x} )}{g( {x - {m\;\Delta\; x}} )}}}}} & (4)\end{matrix}$Linear and nonlinear functions can be decomposed into sums of Gaussiansby eqn. 4 with minimal error introduced (FIG. 15). Further, the Gaborexpansion can be used to estimate the coefficients A_(m) needed todescribe f(x) as a sum of modified Gaussians.

If p_(W)(x), a function with Gaussian sides (with decay length, σ) and aplateau of width w instead of a peak, (FIG. 16A) is used, A_(m) can becalculated using eqn. 3 (as if w=0) and replacing g(x) with p_(w)(x) ineqn. 4. Then the resulting sum,

$\begin{matrix}{{f_{pw}(x)} \approx {( {2\;\pi} )^{- \frac{1}{2}}\frac{\Delta\; x}{\sigma}{\sum\limits_{m = {- \infty}}^{\infty}\;{{f( {m\;\Delta\; x} )}{p_{w}(x)}}}}} & (5)\end{matrix}$overestimates the input function f(x) by a factor s(w/σ) proportional tow/σ. (FIG. 16B):f(x)≈s(w/σ)f _(p)(x).  (6)Thus, an arbitrary function can be expanded as a sum of modifiedGaussians, p_(w)(x) (FIG. 16C).

The Gabor formalism maps directly to the method using laser ablation toproduce protein gradients. The function f(x) defines the gradient alonga path. The variables A_(m) and Δx represent the magnitude ofinactivation and the step size of the translation system (thattranslates the laser relative to the protein-coated substrate)respectively. The function p_(w)(x) corresponds to the inactivationprofile of the laser (Modeled in FIG. 16A) of plateau width w, and σdescribes to the Gaussian sides of the inactivated region (FIG. 16A).These values can be experimentally determined for a given protein andsubstrate and laser and translation system. For a patterning system thatproduces ablated regions with sides with estimated Gaussian widths of σ,a step size, Δx, of the translation system can be found to alwayssatisfy the condition σ≦Δx. The second condition for eqn. 2 is satisfiedby choosing a f(x) with feature dimensions greater than Δx.

Dose tests to relate sets of laser parameters to the magnitude A_(m),and extent, w, of inactivation of surface-bound protein to differentdegrees may be performed. The A_(m) is bounded by 0 and 100%. Becausethe exposure dose depends on laser parameters such as the intensity andthe number of passes, two classes of characterizations at fixed aperturedimensions may be performed. First, the laser intensity is heldconstant, and the number of passes is increased incrementally. In thesecond class, the number of passes is fixed, and the laser intensity isvaried. For example, the laser power may be modulated in steps of 1% ofits maximum output. To determine effect of aperture size on the extentand magnitude of inactivation, these tests may be repeated for a rangeaperture dimensions. To make one-dimensional gradient patterns, theaperture dimension, x, is varied parallel to the gradient. Theperpendicular dimension of the aperture is held constant at its maximum.The results of the aperture test is a set of dose-response curves foreach aperture size and a plot of aperture size versus plateau width w.Fitting the sides of the inactivation profile to Gaussians can determinethe σ for each aperture size. The collection of dose-response curves andσ values relate laser settings to the coefficients A_(m), plateau widthw, and p_(w)(x) of eqn. 5.

The procedure to make gradient patterns of protein activity isstraightforward. In one approach, a gradient function f(x), step sizeΔx, and plateau width w is selected, and the coefficients A_(m) iscalculated from eqn. 3. For each A_(m) the laser system can beprogrammed to expose the protein with an aperture size specified by wand a power and number of passes corresponding to as determined by thedose response curves. There may be multiple sets of laser settings thatmatch a given A_(m), and different sets may be tested to determine theoptimal set for the given f(x) and w. The calculation of the A_(m) maybe automated, and the laser system parameters can be loaded from acomputer file or entered manually.

Two-dimensional gradient patterns may be created using theone-dimensional approach sequentially. For a pattern over an area, a setof one-dimensional paths, ordered such that the entire set covers thearea, can be found. The gradient along each path can be expanded usingthe Gabor formalism, and laser parameters to generate the gradient alongthat path may be determined. Two-dimensional gradient patterns may bemade on planar surfaces as well as on surfaces with topographicfeatures, curved surfaces, or other complex surfaces. The surfaces canbe interior or exterior surfaces. A similar approach can be used tocreate higher-dimensional gradient patterns.

While this disclosure has been described as having exemplaryembodiments, this application is intended to cover any variations, uses,or adaptations using the general principles set forth herein. It isenvisioned that those skilled in the art may devise variousmodifications and equivalents without departing from the spirit andscope of the disclosure as recited in the following claims. Further,this application is intended to cover such departures from the presentdisclosure as come within the known or customary practice within the artto which it pertains.

DOCUMENTS CITED

-   Bastiaans, M. J. (1980). Gabor Expansion of a Signal into Gaussian    Elementary Signals. Proceedings of the Ieee, 68(4): 538-539.-   Bell, System Technical Journal, 27, pp. 379-423 & 623-656, July &    October, 1948.-   Berg et al. Langmuir. 2004. 20:1362-8.-   Chen et al., Science. 1997. 276:1425-8.-   de Wolf, D. A. (1989). Gaussian Decomposition of Beams and Other    Functions. Journal of Applied Physics, 65(12): 5166-5169.-   Dike et al., 1999. In vitro Cell Dev. Biol. Anim. 35, 441-448-   Gabor, D. Theory of communication. The Journal of the Institute of    Electrical Engineers, 93(21)(Part III):429-457, January 1946.-   Ingber, 1992. Semin Cancer Biol. 3:57-63.-   Janssen, A. (1981). Gabor Representation of Generalized-Functions.    Journal of Mathematical Analysis and Applications 83(2): 377-394.-   Recknor et al., Biomaterials. 2006 August; 27(22):4098-108.-   Shannon, C. E. (1948), “A Mathematical Theory of Communication”-   Thery et al., Nat. Cell Biol. 2005. 7:947-953-   Thery et al., Proc. Nat. Acad. Sci. USA. 2006. 103:19771-19776

1. A method for patterning one or more biomolecules on a substrate, themethod comprising: coating the substrate with the one or morebiomolecules; applying a focused laser onto the one or morebiomolecules; ablating a portion of the one or more biomolecules withthe laser in a predetermined pattern, the predetermined pattern havingone or more ablated portions and one or more non-ablated portions on thesubstrate, the one or more ablated portions having less than 100% ofbiological function or activity, or biochemical function or activity ofthe one or more biomolecules on the substrate, and the one or morenon-ablated portions having one or more active or functionalbiomolecules of the one or more biomolecules on the substrate.
 2. Themethod of claim 1, wherein the ablating comprises breaking at least onecovalent bond of the one or more biomolecules.
 3. The method of claim 1,wherein ablating comprises ablating a portion of the one or morebiomolecules with the laser in the predetermined pattern by removing atleast one atom from one or more biomolecules and at most 100% of theatoms of one or more biomolecules, wherein the ablating of the one ormore biomolecules does not substantially destroy or damage the one ormore biomolecules or remove the one or more biomolecules to a depth ofmore than 1 nanometer, more than 3 nanometers, more than 5 nanometers,more than 10 nanometers, more than 25 nanometers, more than 50nanometers, more than 100 nanometers, more than 500 nanometers, morethan 1 micrometer, more than 5 micrometers, more than 10 micrometers,more than 50 micrometers, more than 100 micrometers from a point in anarea irradiated by the laser.
 4. The method of claim 1, wherein applyingthe laser further comprises positioning the substrate relative to thelaser in one, two, or three dimensions to form the predetermined patternby translation and/or rotation of the focused laser radiation relativeto the substrate in one, two, or three dimensions or translation and/orrotation of the substrate relative to the focused laser radiation inone, two, or three dimensions.
 5. The method of claim 1, wherein the oneor more biomolecules are selected from the group consisting of proteins,peptides, nucleic acids, drugs, lipids, bioactive polymers, bioactivecompounds, and any combination thereof.
 6. The method of claim 1,wherein the one or more of biomolecules are attached to a particle orcolloid selected from the group consisting of quantum dots,superparamagnetic nanoparticles, dendrimers, glass particles, silicaparticles, liposomes, viruses, phage particles, and any combinationthereof.
 7. The method of claim 1, wherein the substrate is selectedfrom the group consisting of glass, polymeric material, silicon,plastic, rubber, metal, ceramic, quartz, polydimethylsiloxane,polystyrene, and wherein the substrate comprises a flat or non-flatsurface, a surface of a three-dimensional substrate, or a threedimensional substrate selected from the group of coverslips, slides,microfabricated substrates or devices, micro-electro-mechanical systems(MEMS) devices, Petri dishes, flasks, multiwell plates, vascular stents,auditory implants, implantable devices, complex devices, lab-on-a-chipdevices, microfluidic devices, pipettes, capillaries, capillary tubes,tissue culture flasks, biomedical implant, test tube, eppendorf tube,diagnostic assays, biochips, protein/nucleic acid biochip sensors,cell-based sensors, lab-on-a-chip assays, lab-in-a-capillary assays,cell adhesion assays, cell translocation/migration/invasion/chemotaxisassays, or neuronal-guidance assays, and analogous devices and whereinany material that is not substantially destroyed or damaged by thelaser, and any combination thereof, and wherein the any material that isnot substantially destroyed or damaged by the laser comprises materialthat is not substantially destroyed or damaged when laser irradiation ofan area of substrate removes material to a depth of more than 1nanometer, more than 3 nanometer, more than 5 nanometers, more than 10nanometers, more than 25 nanometers, more than 50 nanometers, more than100 nanometers, more than 500 nanometers, more than 1 micrometer, morethan 5 micrometers, more than 10 micrometers, more than 50 micrometers,more than 100 micrometers from a point in an area irradiated by thelaser.
 8. The method of claim 1, wherein coating the substrate comprisesattaching the one or more biomolecules on the substrate by a methodselected from the group consisting of covalently attaching to thesubstrate, adsorbing to the substrate, attaching via a coupling moleculeof varying length, electrostatically attaching to the substrate,hydrophobically attaching to the substrate, sterically attaching to thesubstrate, entropically attaching to the substrate, and any combinationthereof.
 9. The method of claim 1, wherein coating the substratecomprises coating a non-uniform or prepatterned substrate.
 10. Themethod of claim 1, wherein the laser removes at least one of the one ormore biomolecules at a first dose and inactivates at least one of theone or more biomolecules at a second dose, and wherein the second doseis lower than the first dose.
 11. The method of claim 1, whereinablating comprises patterning a portion of the one or more biomoleculesof one type of biomolecule in a combination of two or more types ofbiomolecules.
 12. The method of claim 1, wherein the predeterminedpattern contains a gradient of inactivated biomolecules, a gradient ofremoved biomolecules, or a combination of gradients of inactivated andremoved biomolecules.
 13. The method of claim 1, wherein the substrateis translucent, wherein applying the laser comprises applying the laserthrough an exterior side of the substrate onto an opposing interior sideof the substrate, wherein the interior side of the substrate has the oneor more biomolecules applied thereon, and wherein said molecules arepartially or fully ablated in the predetermined pattern.
 14. The methodof claim 1, further comprising backfilling the one or more ablatedportions with a second one or more biomolecules.
 15. The method of claim1, wherein the one or more biomolecules are in a dry environment,hydrated by a layer of liquid, in an aqueous solvent environment, or ina non-aqueous solvent environment.
 16. A patterned substrate coated witha one or more biomolecules comprising: a pattern obtainable by coatingthe substrate with the one or more biomolecules, applying a laser ontothe one or more of molecules, and ablating a portion of the one or morebiomolecules with the laser in a predetermined pattern, thepredetermined pattern having one or more ablated portions and one ormore non-ablated portions on the substrate, the one or more ablatedportions having less than 100% of biological function or activity, orbiochemical function or activity of the one or more biomolecules on thesubstrate, and the one or more ablated portions having one or moreactive biomolecules of the one or more biomolecules on the substrate.17. The method of claim 1 wherein the ablating comprises applying thefocused laser at one or more doses at one or more pre-defined positionsin a predefined pattern on the substrate coated with one or morebiomolecules, and the one or more doses and the one or more positionsare determined by applying the Gabor expansion to the predeterminedpattern function.
 18. The method of claim 1 wherein the substratecomprises one or more interior surfaces coated with one or morebiomolecules, including the interior surfaces of hoses and tubes,flasks, sealed chambers, microfluidic systems, lab-on-a-chip devices,pipettes, or capillary tubes.
 19. The method of claim 1 wherein one ormore substrates are translated through the laser patterning system usinga conveyor belt or other translation system.
 20. The substrate of claim16, wherein the substrate comprises one or more interior surfaces withan optical path available to the exterior and coated with one or morebiomolecules, including the interior surfaces of hoses and tubes,flasks, sealed chambers, microfluidic systems, lab-on-a-chip devices,pipettes, or capillary tubes.
 21. The method of claim 1 wherein creatingthe predetermined pattern of ablated and non-ablated portion is bytranslation and/or rotation of the focused laser radiation relative tothe substrate in one, two, or three dimensions or by translation and/orrotation of the substrate relative to the focused laser radiation inone, two, or three dimensions.
 22. The method of claim 21 wherein theone or more ablated portions have at least one dimension in the plane ofthe substrate from at least about 0.1 nanometer, at least about 1nanometer, at least about 10 nanometers, at least about 100 nanometers,or at least about 250 nanometers to about 1 meter.
 23. The method ofclaim 21 wherein the one or more non-ablated portions have at least onedimension in the plane of the substrate from about at least 0.1nanometer, at least about 1 nanometer, at least about 10 nanometers, atleast about 100 nanometers, or at least about 250 nanometers to about 1meter.